DESCRIPTION Abstract: Natural products and compounds derived from or inspired by natural products make up a large fraction of drug molecules. Traditional synthesis strategies based on recovery from the natural source and chemical synthesis approaches present many challenges associated with the purity, scale, and complexity of the compounds, contributing to the raising costs and reduced number of new drug molecules. The development of scalable manufacturing platforms for natural product synthesis will address many challenges faced between natural product drug discovery and therapeutic application. The engineering of biosynthetic pathways in microbial hosts represents a newer approach to chemical synthesis with exciting potential. However, current approaches in metabolic pathway engineering require a significant investment in time and resources and do not scale with the complexity and breadth represented in natural product biosynthesis pathways. As such, for many natural products of interest, microbial biosynthesis strategies are currently viewed as impossible. The goal of the proposed project is to develop synthetic biology platforms that will dramatically advance the application of cellular biosynthesis strategies to natural product drug discovery, development, and production. The scale and efficiency of manufacturing processes that can be engineered into microbial systems will be transformed through the development of new approaches that will enable the implementation of key biosynthesis process optimization strategies. In particular, the project will pioneer and apply the following approaches: (i) noninvasive and real-time detection of metabolite levels, (ii) closed loop embedded control of biosynthesis system behavior, (iii) active organelle routing supporting biosynthesis specialization and compartmentalization; and (iv) highthroughput screening methods for discovery of new biosynthetic activities within a microbial chassis. The power of these new approaches will be demonstrated by using them to broadly enable biosynthesis across an important natural products family exhibiting diverse pharmacological activities, the benzylisoquinoline alkaloids (BIAs). Taken together, the proposed research will provide a foundation for next generation biosynthesis platforms supporting the synthesis of many natural products for drug discovery and production research. The unique expertise of the P.I. in developing foundational tools for controlling and programming biological function will be key to the successful execution of the proposed project. The project will direct cutting-edge advances in synthetic biology to transforming the way in which we currently approach the discovery, development, and synthesis of therapeutic molecules.